Mammalian DNA (cytosine-5) methyltransferases and their expression

Two classes of functional DNA (cytosine-5) methyltransferases have been discovered in mammals to date. One class methylates the unmodified DNA and is designated as the de novo enzyme, whereas the other maintains the methylation status of the daughter strand during DNA replication and thus is referred to as a maintenance DNA methyltransferase. Each enzyme catalyzes methyl group transfer from S-adenosyl-L-methionine to cytosine bases in DNA. During methylation the enzyme flips its target base out of the DNA duplex into a typically concave catalytic pocket. This flipped cytosine base is then a substrate for the enzyme-catalyzed reaction. The newly formed 5-methylcytosine confers epigenetic information on the parental genome without altering nucleotide sequences. This epigenetic information is inherited during DNA replication and cell division. In mammals, DNA methylation participates in gene expression, protection of the genome against selfish DNA, parental imprinting, mammalian X chromosome inactivation, developmental regulation, T cell development, and various diseases.     

Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells.     

The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease

Only one human disease that involves Mendelian inheritance of immunodeficiency and aberrant DNA methylation has been identified. This is a rare chromosome breakage disease called the immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF). Its diagnostic characteristics are agammaglobulinemia with B cells as well as DNA rearrangements targeted to the centromere-adjacent heterochromatic region (qh) of chromosomes 1, 16, and sometimes 9 in mitogen-stimulated lymphocytes. These rearrangement-prone regions show DNA hypomethylation in all examined ICF cell populations. This review summarizes our knowledge about the immunological symptoms of ICF; the nature of DNMT3B mutations in ICF patients; the phenotypes of DNA hypomethylation mutants in humans, mice, and Arabidopsis; the epigenetics of ICF; and ICF-specific RNA expression and cell-surface antigen expression in lymphoblastoid cell lines. Comparisons of ICF and control lymphoblastoid cell lines and ICF patients' symptoms suggest an involvement of DNA methylation in the late stages of lymphocyte maturation.     

The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation

Lipid rafts play an important role in signal integration and cellular activation by the T-cell antigen receptor (TCR). We demonstrate that flotillin-1 and flotillin-2 are important structural raft components, which redistribute to the site of TCR engagement. An antibody to flotillin-1 was able to immobilize other TCR-associated raft components. Although rafts purified from unstimulated cells demonstrated abundant Lck but inabundant LAT, rafts from stimulated cells include an abundance of both components. This suggests dynamic changes in lipid raft composition during CD3/CD28 costimulation. Stimulation of primary human CD4(+) T cells leads to increased GM1 and flotillin-1 expression in the surface membrane, where these components colocalize. This may reconstitute new signaling complexes required for T-cell activation. Altered lipid raft composition and function may play a role in the decline of antigen responsiveness in senescent T cells. In this regard, we observed an increase in the raft-associated gangliolipid, GM1, in resting human CD4(+) and CD8(+) lymphocytes with aging.     

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.     

Quantitative and qualitative assessment of circulating NY-ESO-1 specific CD4+ T cells in cancer-free individuals

The germ cell antigen NY-ESO-1 is characterized by its frequent expression in patients bearing cancers of various histological types, that positively correlates with stage of disease, together with its frequent spontaneous immunogenicity in patients with advanced cancer. Because of these features, NY-ESO-1 is presently viewed as a prototype antigen for the development of cancer vaccines aimed at preventing disease progression. To gain a global view of the CD4+ T cell repertoire available for NY-ESO-1 in individuals of different genetic background, in this study, we have addressed the presence, frequency, and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences among circulating lymphocytes from healthy donors. NY-ESO-1 specific CD4+ T cells were present among circulating lymphocytes at a frequency between 0.5 and 5 precursors per million CD4+ T cells. In the majority of the cases, the reactivity of NY-ESO-1 specific CD4+ T cells was directed towards immunodominant regions located in the carboxyl-terminal half of the protein. Interestingly, immunodominant regions were confined to parts of the NY-ESO-1 protein containing hotspot sequences with predicted high binding for multiple frequently expressed MHC class II molecules. In contrast, no reactivity was found against the amino-terminal part of the protein, which was concomitant with the paucity, in this region, of sequences with predicted high binding to MHC class II molecules.     

Skin-versus gut-skewed homing receptor expression and intrinsic CCR4 expression on human peripheral blood CD4+CD25+ suppressor T cells

In humans and rodents a population of naturally occurring CD4(+)CD25(+) T cells are suppressor T (CD25(+) Ts) cells, which are considered to maintain peripheral immunological tolerance. Recently, we have described a unique chemotactic response profile for human CD25(+) Ts cells, but their homing potential remains poorly defined. Here, we document a heterogeneous homing potential of human peripheral blood CD25(+) Ts cells consistent with their ability to mediate immunosuppression at distinct locations. Surprisingly, CD25(+)Ts cells are depleted of gut-homing integrin alpha(4) (+)beta(7) (+) T cells, while being enriched in skin-homing cutaneous lymphocyte antigen (CLA)(+) T cells. These findings document heterogeneous homing potential of peripheral blood-borne CD25(+) Ts cells with marked skewing for skin- versus gut-homing. Expression of CCR4 associates with both CD25 and CLA cell surface markers, being highest on CD4(+)CLA(+)CD25(+) T cells. Importantly, CD4(+)CD25(+) Ts cells isolated from human cord blood lack expression of CLA while expressing CCR4, suggesting intrinsic expression of CCR4 on CD25(+) Ts cells. These observations indicate that the increased expression of CCR4, which is proposed to guide CD25(+) Ts cells to DC, is an intrinsic feature of CD25(+) Ts cells.     

Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis

As a tumour necrosis factor receptor superfamily member, 4-1BB (CD137) is preferentially expressed in CD4+CD25+ regulatory T cells (Tregs) and has been suggested to play an important role in regulating the generation or function of Tregs. Recent studies of human Tregs have shown that blood CD4+CD25(high) T cells were much closer to Tregs in terms of their functionality. Furthermore, CD4+CD25(high) Tregs have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we examined the expression of 4-1BB and soluble 4-1BB (s4-1BB) protein levels in the peripheral blood of MS patients. Compared with healthy controls, MS patients had decreased 4-1BB expression in their CD4+C25(high) Tregs and increased plasma s4-1BB protein levels. Moreover, the plasma s4-1BB levels of MS patients were shown to be inversely correlated with the 4-1BB surface expression of CD4+CD25(high) Tregs. The down-regulated 4-1BB expression on CD4+CD25(high) Tregs of MS patients may be involved in the impaired immunoactivity of these Tregs. The elevated s4-1BB levels may, at least in part, function as a self-regulatory attempt to inhibit antigen-driven proliferation of Tregs or their immunosuppressive activity.     

The morphology and distribution of CD1a positive Langerhans cells in normal and squamous cell carcinoma of cervix

Introduction

Langerhans cells (LCs), a type of dendritic cells are the professional antigen presenting cells present in the mucosa surfaces. They play an important role in antitumor immune response. The present study aims to find out the morphology and distribution of CD1a positive LCs in normal and squamous cell carcinoma of cervix.

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation

Activation of naive CD4⁺ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4⁺ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4⁺ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4⁺ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4⁺ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.     

Idiopathic CD4+ T cell lymphocytopenia with the absence of B cells and CD8+28+ cells in peripheral blood

The absence of B cells and a severe decrease in CD8+28+ cells were observed in two female children with CD4+ T cell lymphocytopenia. Idiopathic (primary) CD4+ lymphocytopenia is a rare entity and its pathogenesis and genetics are not yet known. The literature was reviewed, in particular for severe alterations in B and CD8+28+ cells and for the role of NF-kappa B and p56^lck in the immunopathogenesis. Whether the underlying mechanism in idiopathic CD4+ lymphocytopenia is found or not, these patients who present with severe symptoms of a combined immunodeficiency must be treated with intravenous immunoglobin regularly until they have a compatible donor for bone marrow transplantation. Received: 4 May 2001 / Accepted: 10 October 2002 Correspondence to N. Kutukculer     

DNA甲基化在急性冠脉综合征患者CD4+CD28-T细胞CD28表达缺失中的作用

目的 通过研究急性冠脉综合征(acute coronary syndrome,ACS)患者外周血CD4+T细胞CD28 mRNA水平和CD28基因启动子调节序列的甲基化状态,旨在探讨DNA甲基化在ACS患者CD4+CD28-T细胞CD28表达缺失中的作用.方法 免疫磁珠分离CD4+T细胞经逆转录后,实时定量PCR(real time-PCR)技术检测CD4+T细胞CD28mRNA的表达水平,亚硫酸氢钠测序检测CD28基因启动子调节序列的甲基化状态.结果 与正常对照组相比,ACS患者CD4+T细胞CD28mRNA表达水平显著减低,差异具有统计学意义[正常对照组比ACS组:(1.066±0.162)比(0.401±0.069),P<0.05].CD28基因启动子区域的甲基化水平显著增高,差异有统计学意义[正常对照组比ACS组:(24.47±3.17)%比(43.33±1.52)%,P<0.05].CD28基因启动子区域DNA甲基化水平与CD28mRNA表达水平呈显著负相关(P=0.01,r=-0.579).结论 ACS患者CD4+T细胞CD28基因启动子区域高甲基化调控了CD28基因转录抑制.DNA甲基化参与了CD4+CD28-T细胞的形成.     

Arterial blood gas control in the upright versus recumbent Asian elephant

In the elephant, there is concern that lateral recumbency (LR) impairs respiratory muscle and lung function resulting in clinically significant arterial hypoxemia. Using healthy adult female Asian elephants (Elephas maximus, n=6), the hypothesis was tested that, given the O(2) binding characteristics of elephant blood, substantial reductions in arterial O(2) pressure (Pa(O(2))) in LR could be tolerated without lowering arterial O(2) content appreciably. Fifteen minutes of LR decreased Pa(O(2)) from 103+/-2 (upright, U) to 77+/-4 mmHg (P<0.05) and hemoglobin O(2) saturation (U, 97.8+/-0.1, LR, 95.3+/-0.5%, P<0.05). However, due to a recumbency-induced hemoconcentration, arterial O(2) content was unchanged (U, 18.2+/-2.4, LR, 18.3+/-2.1 ml O(2) per 100 ml). In addition, there was a mild hyperventilation in LR that reduced arterial CO(2) pressure (P(CO(2))) from 39.4+/-0.3 to 37.1+/-1.0 mmHg (P<0.05). These data indicate that the Asian elephant can endure at least short periods of LR without lowering arterial O(2) content.     

慢性乙型肝炎患者外周血细胞CD40+、CD40L+及CD8+/CD28+表达的研究

目的检测慢性乙型肝炎(简称慢乙肝)患者外周血淋巴、单核细胞表面CD40+、CD40L+及淋巴细胞表面CD8+/CD28+、CD8+/CD28-的表达,评价患者的细胞免疫状态,为临床治疗提供指导.方法流式细胞仪测定慢乙肝患者单核细胞、淋巴细胞表面CD40+、CD40L+表达的百分率及淋巴细胞表面CD8+/CD28+、CD8+/CD28-表达的百分率.结果慢乙肝组外周血淋巴、单核细胞表面CD40+、CD40L+及淋巴细胞表面CD8+/CD28+的表达明显低于正常对照组(P＜0.01,P＜0.05,P＜0.01,P＜0.05,P＜0.01),乙肝肝硬化组(简称肝硬化)均明显低于正常对照组(均P＜0.01),而慢乙肝组、肝硬化组CD8+/CD28-的表达高于正常对照组(P＜0.05,P＜0.01).慢乙肝组与肝硬化组均无显著差异(均P＞0.05).慢乙肝轻、中、重度和肝硬化三组间均无显著差异(均P＞0.05).相关性分析结果显示,慢乙肝患者淋巴、单核细胞表面CD40+和CD40L+的表达之间存在正相关,淋巴细胞CD40+、CD40L+表达与CD8+/CD28+表达存在正相关,而与CD8+/CD28-表达相关性不明显.结论慢乙肝患者外周血淋巴、单核细胞CD40+、CD40L+及淋巴细胞CD8+/CD28+表达低下,而CD8+/CD28-的表达增加.检测外周血CD40+、CD40L+及CD8+/CD28+的表达可评估患者的细胞免疫状态,对临床的抗病毒治疗提供新思路.     

Expansion of circulating NKG2D+ effector memory T-cells and expression of NKG2D-ligand MIC in granulomaous lesions in Wegener's granulomatosis

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.     

Peripheral blood and granuloma CD4(+)CD28(-) T cells are a major source of interferon-gamma and tumor necrosis factor-alpha in Wegener's granulomatosis

To elucidate whether the fraction of CD28(-) T cells within the CD4(+) T-cell population is a major source of Th1-like and proinflammatory cytokine production driving Wegener's granulomatosis (WG) granuloma formation, we analyzed the phenotype and functional characteristics of peripheral blood CD4(+)CD28(-) T cells and of T cells in granulomatous lesions of 12 patients with active WG. Surface markers and intracytoplasmic cytokine and perforin expression were assessed by flow cytometry. Cytokine secretion was measured by enzyme-linked immunosorbent assay. Immunohistological studies demonstrated interferon-gamma and tumor necrosis factor-alpha cytokine positivity attributable to CD4(+)CD28(-) T cells in granulomatous lesions. Peripheral blood CD4(+)CD28(-) T cells expressed CD57, also found on natural killer cells, and intracytoplasmic perforin. They were generally CD25 (interleukin-2 receptor)-negative. CD18 (adhesion molecule beta(2)-integrin) was strongly up-regulated on CD4(+)CD28(-) T cells, whereas only a minority of CD4(+)CD28(+) T cells expressed CD18. CD4(+)CD28(-) T cells appeared as a major source of interferon-gamma and tumor necrosis factor-alpha. In contrast, CD4(+)CD28(+) T cells were able to produce and secrete a wider variety of cytokines including interleukin-2. One-quarter of CD4(+)CD28(+) T cells expressed the activation marker CD25, but they lacked perforin. Thus, CD4(+)CD28(-) T cells appeared more differentiated than CD4(+)CD28(+) T cells. They displayed Th1-like cytokine production and features suggestive of the capability of CD4(+) T-cell-mediated cytotoxicity. CD4(+)CD28(-) T cells may be recruited into granulomatous lesions from the blood via CD18 interaction, and may subsequently promote monocyte accumulation and granuloma formation through their cytokine secretion in WG.     

ISSag1 in streptococcal strains of human and animal origin

The chromosomal region of Streptococcus agalactiae harboring the C5a peptidase and the lmb genes displays the structure of a composite transposon. Its presence in a streptococcal strain is associated with the origin of this strain from a human host. In S. agalactiae it is flanked by two copies of the insertion element ISSag2, and the nucleotide sequence for a third IS element (ISSag1) can be found in this region. Based on amino acid sequence similarity of the deduced transposase ISSag1 belongs to the IS3 family. It is 1251 bp long and flanked by 37 bp imperfect inverted repeats. Horizontal gene transfer among different bacterial species is facilitated by mobile genetic elements. To investigate if ISSag1 homologues are also present in other streptococcal species, various species of pyogenic streptococci from animal and human origin were analyzed by Southern blot hybridization and PCR. Among the different streptococcal species, multiple copies of an ISSag1 homologue could only be detected in S. dysgalactiae subsp. dysgalactiae strains of animal origin. All of the S. agalactiae strains harbored only a single copy, that was always found in strains with the scpB-lmb composite transposon. A single copy of an ISSag1 homologue could also be detected in some of the S. pyogenes and S. dysgalactiae subsp. equisimilis strains. Nucleotide sequencing of the IS element in S. dysgalactiae subsp. dysgalactiae strains revealed several different variants. One of the variants showed the features of a regular IS3 element. The other two variants that were observed displayed a 500-bp deletion and a mosaic structure composed of ISSag1 and ISSag2 homologues. This mosaic structure suggests that recombination and horizontal gene transfer events in S. dysgalactiae strains of bovine origin could have played a role in the assembly of the scpB-lmb composite transposon structure.